Complex I (NADH:quinone oxidoreductase) is the entry point of NADH into the electron transport chain of mitochondria. It oxidizes NADH to NAD+ which allows the citric acid cycle to oxidize carbon compounds. Its extraction of two electrons from NADH is used to reduce ubiquinone to ubiquinol, which then functions as an electron carrier to downstream enzymes. This reduction is coupled to the translocation of protons across the membrane, by an unknown mechanism. This generates the electrochemical proton gradient that is used to synthesize ATP. In humans Complex I plays additional roles in the mitochondrion related to oxidative stress, cell signaling, and cell death. For example, blockage of electron transport can increase the production of superoxide by Complex I, leading to oxidative damage of DNA, lipids, and protein. Loss of Complex I function is associated with the onset of idiopathic Parkinson's disease. Complex I subunits are targeted by proteases that trigger cell death processes. Mutations in the protein subunits of Complex I are risk factors for a wide range of diseases and disorders, such as multiple sclerosis, Type 2 diabetes, and bipolar disorder. Some such alleles are known to be causative for fatal disorders such as Leber's hereditary optic neuropathy (LHON), Leigh disease, and mitochondrial encephalomyopathy and lactic acidosis with stroke-like episodes (MELAS). It has been difficult to understand the effect of mitochondrial mutations in Complex I because so little is known about the assembly and function of its membrane subunits. Recent structural studies of bacterial Complex I have revealed new details about the membrane subunits. The objectives of this project are to test the hypothesis that an unusually long alpha-helix, positioned laterally along the membrane, is a key component of proton translocation. A bacterial model system will be used to carry out these studies because of the ability to construct mutations in the hydrophobic proteins that carry out proton translocation. The specific aims of this proposal are first to determine if a functional Complex I can be assembled with a truncated L subunit- one which lacks some or all of its carboxy terminal extension that has been proposed to act as a piston in conformational coupling. Since the los of this region of subunit L might prevent assembly, so two additional aims are planned. Second, the importance of piston-like motion will be tested by constructing cysteine residues in subunit L as sites for photo-affinity cross-linking to other subunits. And third, to test whether the lateral helix needs to be intact for function, protease sites will be constructed at several locations, allowing cleavage after assembly of the enzyme. From the results of this research it will be possible to learn if piston-like motion by subunit L is necessary for proton translocation, which will give insight into many of the diseases associated with the membrane subunits of Complex I. PUBLIC HEALTH RELEVANCE: Recent studies have revealed that mitochondria not only are the energy production centers of cells, but also are key players in the control of cell death. Mitochondria contain a small subset of the human genome, and several of these genes code for proteins that are components of an extremely important enzyme called Complex I. This enzyme is both a target and an initiator of cell death, and its polymorphisms are risk factors or causative agents for many degenerative diseases. The work in described in this proposal will allow greater understanding of key aspects of the functioning of Complex I.